IP Library › Granted Patent US 12,378,537
Granted Patent B2
US 12,378,537 · App. 17/379,843 · Granted Aug 5, 2025

Biocatalysts and methods for synthesizing derivatives of tryptamine and tryptamine analogs

Inventors: Jovana Nazor (Milpitas, CA); Derek J. Smith (Singapore, SG); Michael A. Crowe (Singapore, SG); Shiwei Song (Singapore, SG); Steven J. Collier (Concord, MA)
Assignee: Codexis, Inc.
C12N9/1096C12P17/10C12Y206/01C12Y206/01018Y02P20/52
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Quick Facts
Patent No.
US 12,378,537
App. No.
17/379,843
Granted
Aug 5, 2025
Kind
B2
Abstract

The present disclosure provides engineered transaminase polypeptides for the production of amines, polynucleotides encoding the engineered transaminases, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases to prepare compounds useful in the production of active pharmaceutical agents.

Claims (105)

1. An engineered polypeptide having transaminase activity, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4, and a residue difference X284A/G as compared to SEQ ID NO: 4.

2. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises one or more residue differences as compared to SEQ ID NO: 4 at residue positions selected from: X26, X31, X33, X41, X47, X57, X70, X86, X88, X107, X132, X148, X163, X168, X173, X203, X250, X314, X324, X346, X395, X398, X400, X417, X419, X423, X448, and X451.

3. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises at least one or more residue differences selected from X26R, X31S/D, X86D, X163I/L/R/V, X315G, X398L/V/W, and X400G.

4. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises a combination of residue differences selected from:

X14V and X163I/L/R/V,

X86D and X400G,

X57F/Y and X163I/L/R/V,

X57F/Y and X398L/V/W,

X14V, X113L/V, X163I/L/R/V, and X424V,

X31S, X57F/Y, X163I/L/R/V, X315G, X346L, and X398L/V/W

X14V, X113L, X163L, and X424V,

X14V, X26R, X163L, and X400G,

X14V, X26R, X88L, and X113L,

X57F, X163L, X168K, X314N, X315G, X346L, and X398V,

X14V, X163L, X173A, X400G, and X420N,

X14V, X113L, and X163L,

X14V, X26R, X163L, and X400G, and

X14V, X33T, X57F, X113L, and X163L.

5. The engineered polypeptide of claim 1 , wherein said polypeptide has at least 1.2 fold increased stability as compared to the polypeptide of SEQ ID NO:4.

6. The engineered polypeptide of claim 1 , wherein the amino acid sequence comprises SEQ ID NO: 134.

7. The engineered polypeptide of claim 1 , wherein said polypeptide is immobilized on a solid support.

8. A process for preparing a compound of formula (I),

wherein

R 1 is selected from the group consisting of hydrogen, carboxy, carboxy(C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyl, optionally substituted (C 2 -C 6 )alkenyl, optionally substituted alkyloxycarbonyl, optionally substituted arylcarbonyl, optionally substituted arylsulfonyl, and a protecting group;

R 2 is selected from the group consisting of hydrogen, oxo, halo, hydroxy, amino, thio, optionally substituted (C 1 -C 6 )alkyl, (C 1 -C 6 )alkylamino, (C 1 -C 6 )dialkylamino, (C 1 -C 6 )alkylthio, (C 1 -C 6 )alkylsulfonyl, (C 1 -C 6 )alkylsulfinyl, and optionally substituted (C 1 -C 6 )alkyloxy;

R 4 , R 6 and R 7 are each, independently of the others, selected from the group consisting of hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C 1 -C 6 )alkyl, hydroxy(C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyloxy, optionally substituted (C 1 -C 6 )alkylamino, optionally substituted (C 1 -C 6 )dialkylamino, optionally substituted (C 1 -C 6 )alkylthio, optionally substituted (C 1 -C 6 )alkylsulfonyl, optionally substituted (C 1 -C 6 )alkylsulfinyl, carboxy(C 1 -C 6 )alkyl, (C 1 -C 6 )alkyloxycarbonyl, (C 1 -C 6 )alkylcarbonyloxy, optionally substituted aminocarbonyl, aminocarbonyl (C 1 -C 6 )alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted arylthio, optionally substituted arylsulfonyl, optionally substituted arylsulfinyl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyloxy, optionally substituted heteroaryloxy, optionally substituted heteroarylamino, optionally substituted heteroarylthio, optionally substituted heteroarylsulfonyl, optionally substituted heteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyloxy, alkylaminosulfonyl(C 1 -C 6 )alkyl, arylsulfonyl(C 1 -C 6 )alkyl, and heteroarylsulfonyl(C 1 -C 6 )alkyl;

R 5 is selected from the group consisting of hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C 1 -C 6 )alkyl, hydroxy(C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyloxy, optionally substituted (C 1 -C 6 )alkylamino, optionally substituted (C 1 -C 6 )dialkylamino, optionally substituted (C 1 -C 6 )alkylthio, optionally substituted (C 1 -C 6 )alkylsulfonyl, optionally substituted (C 1 -C 6 )alkylsulfinyl, carboxy(C 1 -C 6 )alkyl, (C 1 -C 6 )alkyloxycarbonyl, (C 1 -C 6 )alkylcarbonyloxy, optionally substituted aminocarbonyl, aminocarbonyl (C 1 -C 6 )alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted arylthio, optionally substituted arylsulfonyl, optionally substituted arylsulfinyl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyloxy, optionally substituted heteroaryloxy, optionally substituted heteroarylamino, optionally substituted heteroarylthio, optionally substituted heteroarylsulfonyl, optionally substituted heteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyloxy, alkylaminosulfonyl(C 1 -C 6 )alkyl, arylsulfonyl(C 1 -C 6 )alkyl, and heteroarylsulfonyl(C 1 -C 6 )alkyl, or together with R 4 forms a 5 to 8 membered optionally substituted cycloalkyl or optionally substituted heterocyclic ring;

R 8 is selected from the group consisting of optionally substituted (C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyloxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl, or together with R 2 forms an optionally substituted 5 to 8 membered cycloalkyl or heterocyclic ring; and

n is 1 or 2;

with the provisos that

(a) when R 2 is hydrogen, then at least one of the following applies:

(i) R 1 is not hydrogen, methyl, 4-(methyloxy)phenylcarbonyl-, 4-(trifluoromethyloxy)phenylsulfonyl-, 3-bromophenylcarbonyl-, 3-aminopropyl-, or 3-(methylcarbonylamino)propyl-;

(ii) R 4 and R 7 are each, independently of the other, not hydrogen or chloro;

(iii) R 5 is not hydrogen, hydroxy, methyl, methyloxy, fluoro, chloro, trifluoromethyl, or cyano;

(iv) R 6 is not hydrogen, hydroxy, methyloxy, fluoro or chloro; or

(v) R 8 is not methyl, ethyl, hydroxymethyl, or trifluoromethyl-; and

(b) when n is 1, R 2 and R 8 together form a cyclohexyl ring, and R 1 , R 4 , R 6 , and R 7 are hydrogen, then R 5 is not fluoro;

comprising contacting the substrate compound of formula (II),

wherein R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 and n are as defined above,

in presence of an amino donor under suitable reaction conditions with an engineered polypeptide of claim 1 .

9. The process of claim 8 , wherein the compound of formula (I) comprises the compound of formula (IS),

having the indicated stereochemistry at the carbon atom marked with an *, wherein compound (IS) is formed in enantiomeric excess.

10. A process for preparing a compound of formula (Ia):

wherein,

R 1 is selected from the group consisting of hydrogen, carboxy, carboxy(C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyl, optionally substituted (C 2 -C 6 )alkenyl, optionally substituted alkyloxycarbonyl, optionally substituted arylcarbonyl, optionally substituted arylsulfonyl, and a protecting group;

R 2 is selected from the group consisting of hydrogen, oxo, halo, hydroxy, amino, thio, optionally substituted (C 1 -C 6 )alkyl, (C 1 -C 6 )alkylamino, (C 1 -C 6 )dialkylamino, (C 1 -C 6 )alkylthio, (C 1 -C 6 )alkylsulfonyl, (C 1 -C 6 )alkylsulfinyl, and optionally substituted (C 1 -C 6 )alkyloxy;

R 4 , R 5 , R 6 and R 7 are each, independently of the others, selected from the group consisting of hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C 1 -C 6 )alkyl, hydroxy(C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyloxy, optionally substituted (C 1 -C 6 )alkylamino, optionally substituted (C 1 -C 6 )dialkylamino, optionally substituted (C 1 -C 6 )alkylthio, optionally substituted (C 1 -C 6 )alkylsulfonyl, optionally substituted (C 1 -C 6 )alkylsulfinyl, carboxy(C 1 -C 6 )alkyl, (C 1 -C 6 )alkyloxycarbonyl, (C 1 -C 6 )alkylcarbonyloxy, optionally substituted aminocarbonyl, aminocarbonyl (C 1 -C 6 )alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted arylthio, optionally substituted arylsulfonyl, optionally substituted arylsulfinyl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyloxy, optionally substituted heteroaryloxy, optionally substituted heteroarylamino, optionally substituted heteroarylthio, optionally substituted heteroarylsulfonyl, optionally substituted heteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyloxy, alkylaminosulfonyl(C 1 -C 6 )alkyl, arylsulfonyl(C 1 -C 6 )alkyl, and heteroarylsulfonyl(C 1 -C 6 )alkyl; and

R 8 is selected from the group consisting of optionally substituted (C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyloxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl;

with the proviso that when R 2 is hydrogen, then at least one of the following applies:

(i) R 1 is not hydrogen, methyl, 4-(methyloxy)phenylcarbonyl-, 4-(trifluoromethyloxy)phenylsulfonyl-, 3-bromophenylcarbonyl-, 3-aminopropyl-, or 3-(methylcarbonylamino)propyl-;

(ii) R 4 and R 7 are each, independently of the other, not hydrogen or chloro;

(iii) R 5 is not hydrogen, hydroxy, methyl, methyloxy, fluoro, chloro, trifluoromethyl or cyano;

(iv) R 6 is not hydrogen, hydroxy, methyloxy, fluoro or chloro; or

(v) R 8 is not methyl, ethyl, hydroxymethyl-, or trifluoromethyl-;

comprising contacting the substrate compound of formula (IIa)

wherein R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , and R 8 are as defined above,

in presence of an amino donor under suitable reaction conditions with an engineered transaminase polypeptide of claim 1 .

11. The process of claim 10 , wherein the compound of formula (Ia) comprises the compound of formula (IaS)

having the indicated stereochemistry at the carbon atom marked with an *, wherein compound (IaS) is formed in enantiomeric excess.

12. A process for preparing a compound of formula (Ia1)

wherein

R 1 is selected from the group consisting of hydrogen and (C 1 -C 6 )alkyl;

R 2 is selected from the group consisting of hydrogen, halo, and (C 1 -C 6 )alkyl;

R 4 , R 5 , R 6 , and R 7 are each, independently of the others, selected from the group consisting of hydrogen, halo, hydroxy, optionally substituted (C 1 -C 6 )alkyl, hydroxy(C 1 -C 6 )alkyl, (C 1 -C 6 )alkyl; amino, (C 1 -C 6 )alkylamino, and (C 1 -C 6 )dialkylamino; and

R 9 is selected from the group consisting of halo, hydroxy, hydroxy(C 1 -C 6 )alkyl, amino, (C 1 -C 6 )alkylamino, and (C 1 -C 6 )dialkylamino;

with the proviso that when R 2 is hydrogen, then at least one of the following applies:

(i) R 1 is not hydrogen or methyl;

(ii) R 4 and R 7 are each, independently of the other, not hydrogen or chloro;

(iii) R 5 is not hydrogen, hydroxy, methyl, methyloxy, fluoro, chloro, or trifluoromethyl;

(iv) R 6 is not hydrogen, hydroxy, methyloxy, fluoro or chloro; or

(v) R 9 is not hydroxy;

comprising contacting the substrate compound of formula (IIa1)

wherein

R 1 , R 2 , R 4 , R 5 , R 6 , R 7 and R 9 are as defined above,

in presence of an amino donor under suitable reaction conditions with an engineered polypeptide of claim 1 .

13. A process for preparing a compound of formula (Ia2)

wherein

L is a leaving group;

each occurrence of R 10 is, independently of the others, selected from the group consisting of halo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C 1 -C 6 )alkyl, hydroxy(C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyloxy, optionally substituted (C 1 -C 6 )alkylamino, optionally substituted (C 1 -C 6 )dialkylamino, optionally substituted (C 1 -C 6 )alkylthio, optionally substituted (C 1 -C 6 )alkylsulfonyl, optionally substituted (C 1 -C 6 )alkylsulfinyl, carboxy(C 1 -C 6 )alkyl, (C 1 -C 6 )alkyloxycarbonyl, (C 1 -C 6 )alkylcarbonyloxy, optionally substituted aminocarbonyl, aminocarbonyl (C 1 -C 6 )alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted arylthio, optionally substituted arylsulfonyl, optionally substituted arylsulfinyl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyloxy, optionally substituted heteroaryloxy, optionally substituted heteroarylamino, optionally substituted heteroarylthio, optionally substituted heteroarylsulfonyl, optionally substituted heteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyloxy, alkylaminosulfonyl(C 1 -C 6 )alkyl, arylsulfonyl(C 1 -C 6 )alkyl, and heteroarylsulfonyl(C 1 -C 6 )alkyl;

k is an integer from 3 to 5;

n is 1 or 2; and

w is an integer from 0 to 4;

comprising contacting the substrate compound of formula (IIa2)

wherein

L, R 10 , k, n and w are as defined above;

in presence of an amino donor under suitable reaction conditions with an engineered polypeptide of claim 1 .

14. The process of claim 13 , wherein L is Cl or Br and k is 3.

15. A process for preparing a compound of formula (Ib)

wherein

Z is selected from the group consisting of O, S, NH or —(CH 2 ) m —, wherein m is 0, 1, 2 or 3;

R 1 is selected from the group consisting of hydrogen, carboxy, carboxy(C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyl, optionally substituted (C 2 -C 6 )alkenyl, optionally substituted alkyloxycarbonyl, optionally substituted arylcarbonyl, optionally substituted arylsulfonyl, and a protecting group;

R 4 , R 5 , R 6 and R 7 are each, independently of the others, selected from the group consisting of hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C 1 -C 6 )alkyl, hydroxy(C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyloxy, optionally substituted (C 1 -C 6 )alkylamino, optionally substituted (C 1 -C 6 )dialkylamino, optionally substituted (C 1 -C 6 )alkylthio, optionally substituted (C 1 -C 6 )alkylsulfonyl, optionally substituted (C 1 -C 6 )alkylsulfinyl, carboxy(C 1 -C 6 )alkyl, (C 1 -C 6 )alkyloxycarbonyl, (C 1 -C 6 )alkylcarbonyloxy, optionally substituted aminocarbonyl, aminocarbonyl (C 1 -C 6 )alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted arylthio, optionally substituted arylsulfonyl, optionally substituted arylsulfinyl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyloxy, optionally substituted heteroaryloxy, optionally substituted heteroarylamino, optionally substituted heteroarylthio, optionally substituted heteroarylsulfonyl, optionally substituted heteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyloxy, alkylaminosulfonyl(C 1 -C 6 )alkyl, arylsulfonyl(C 1 -C 6 )alkyl, and heteroarylsulfonyl(C 1 -C 6 )alkyl;

with the proviso that when Z is —(CH 2 ) m —, m is 1, and R 1 , R 4 , R 6 and R 7 are hydrogen, then R 5 is not fluoro;

comprising contacting the substrate compound of formula (IIb)

wherein Z, R 1 , R 4 , R 5 , R 6 , and R 7 are as defined above;

in presence of an amino donor under suitable reaction conditions with an engineered polypeptide of claim 1 .

16. A process for preparing a compound of formula (Ib1)

wherein

R 1 is selected from the group consisting of hydrogen, carboxy(C 1 -C 6 )alkyl, and (C 1 -C 6 )alkyl;

each occurrence of R 11 is, independently of the others, selected from the group consisting of chloro, bromo, iodo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C 1 -C 6 )alkyl, hydroxy(C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkyloxy, optionally substituted (C 1 -C 6 )alkylamino, optionally substituted (C 1 -C 6 )dialkylamino, optionally substituted (C 1 -C 6 )alkylthio, optionally substituted (C 1 -C 6 )alkylsulfonyl, optionally substituted (C 1 -C 6 )alkylsulfinyl, carboxy(C 1 -C 6 )alkyl, (C 1 -C 6 )alkyloxycarbonyl, (C 1 -C 6 )alkylcarbonyloxy, optionally substituted aminocarbonyl, aminocarbonyl (C 1 -C 6 )alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted arylthio, optionally substituted arylsulfonyl, optionally substituted arylsulfinyl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyloxy, optionally substituted heteroaryloxy, optionally substituted heteroarylamino, optionally substituted heteroarylthio, optionally substituted heteroarylsulfonyl, optionally substituted heteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyloxy, alkylaminosulfonyl(C 1 -C 6 )alkyl, arylsulfonyl(C 1 -C 6 )alkyl, and heteroarylsulfonyl(C 1 -C 6 )alkyl; and

w is an integer from 0 to 4;

comprising contacting the substrate compound of formula (IIb1)

wherein

R 1 , R 11 and w are as defined above;

in presence of an amino donor under suitable reaction conditions with an engineered polypeptide of claim 1 .

Assignments (1)
SECURITY INTEREST Recorded Feb 15, 2024
From: CODEXIS, INC.
To: INNOVATUS LIFE SCIENCES LENDING FUND I, LP, AS COLLATERAL AGENT
Reel/Frame 066600/0650 →
Continuity (7)
Continuation 16707724 · Dec 9, 2019
Continuation 15623698 · Jun 15, 2017
Continuation 15180355 · Jun 13, 2016
Division 14828839 · Aug 18, 2015
Division 14386082
Provisional Application 61614666 · Mar 23, 2012
Related Publication 20210348139A1 · Nov 11, 2021
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